U.S. patent number 6,099,848 [Application Number 08/972,902] was granted by the patent office on 2000-08-08 for immunogenic compositions comprising dal/dat double-mutant, auxotrophic, attenuated strains of listeria and their methods of use.
This patent grant is currently assigned to The Trustees of the University of Pennsylvania. Invention is credited to Fred R. Frankel, Daniel A. Portnoy.
United States Patent |
6,099,848 |
Frankel , et al. |
August 8, 2000 |
Immunogenic compositions comprising DAL/DAT double-mutant,
auxotrophic, attenuated strains of Listeria and their methods of
use
Abstract
Listeria monocytogenes is an intracellular bacterial pathogen
that elicits a strong cellular immune response following infection
and therefore has potential use as a vaccine vector. However, while
infections by L. monocytogenes are fairly rare and can readily be
controlled by a number of antibiotics, the organism can
nevertheless cause meningitis and death, particularly in
immunocompromised or pregnant patients. We therefore have
endeavored to isolate a highly attenuated strain of this organism
for use as a vaccine vector. D-Alanine is required for the
synthesis of the mucopeptide component of the cell walls of
virtually all bacteria and is found almost exclusively in the
microbial world. We have found in L. monocytogenes two genes that
control the synthesis of this compound, an alanine racemase gene
(dal) and a D-amino acid aminotransferase gene (dat). By
inactivating both genes, we produced an organism that could be
grown in the laboratory when supplemented with D-alanine but was
unable to grow outside the laboratory, particularly in the
cytoplasm of eukaryotic host cells, the natural habitat of this
organism during infection. In mice, the double-mutant strain was
completely attenuated. Nevertheless, it showed the ability,
particularly under conditions of transient suppression of the
mutant phenotype, to induce cytotoxic T-lymphocyte responses and to
generate protective immunity against lethal challenge by wild-type
L. monocytogenes equivalent to that induced by the wild-type
organism.
Inventors: |
Frankel; Fred R. (Philadelphia,
PA), Portnoy; Daniel A. (Albany, CA) |
Assignee: |
The Trustees of the University of
Pennsylvania (Philadelphia, PA)
|
Family
ID: |
25520272 |
Appl.
No.: |
08/972,902 |
Filed: |
November 18, 1997 |
Current U.S.
Class: |
424/246.1;
424/200.1; 435/71.2; 424/93.46; 424/234.1 |
Current CPC
Class: |
C07K
14/195 (20130101); C07K 14/005 (20130101); A61P
37/04 (20180101); A61P 35/00 (20180101); C12N
1/205 (20210501); A61P 31/00 (20180101); C12R
2001/01 (20210501); A61K 39/00 (20130101); C12N
2740/16022 (20130101) |
Current International
Class: |
C07K
14/16 (20060101); C07K 14/195 (20060101); C07K
14/005 (20060101); A61K 39/00 (20060101); A61K
039/07 () |
Field of
Search: |
;435/7.32,71.1,71.2,320.1,243
;424/184.1,200.1,201.1,234.1,93.46,246.1 |
References Cited
[Referenced By]
U.S. Patent Documents
|
|
|
5830702 |
November 1998 |
Portnoy et al. |
|
Other References
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|
Primary Examiner: Scheiner; Laurie
Assistant Examiner: Parkin; J. S.
Attorney, Agent or Firm: Akin, Gump, Strauss, Hauer &
Feld, L.L.P.
Government Interests
GOVERNMENT SUPPORT
This invention was supported in part by funds from the U.S.
Government (NIH Grant Nos. AI-26919 and AI-27655) and the U.S.
Government may therefore have certain rights in the invention.
Claims
What is claimed is:
1. A method of eliciting a T cell immune response to an antigen in
a mammal comprising administering to said mammal an auxotrophic
attenuated strain of Listeria which expresses said antigen, wherein
said auxotrophic attenuated strain comprises a mutation in both the
dal and dat genes in the genome of said Listeria.
2. The method of claim 1, wherein said Listeria is L.
monocytogenes.
3. The method of claim 1, wherein said auxotrophic attenuated
strain further comprises DNA encoding a heterologous antigen.
4. The method of claim 1, wherein said auxotrophic attenuated
strain further comprises a vector comprising a DNA encoding a
heterologous antigen.
5. The method of claim 3, wherein said heterologous antigen is an
HIV-1 antigen.
6. The method of claim 4, wherein said heterologous antigen is an
HIV-1 antigen.
7. An immunogenic composition that is capable of inducing a strong
CTL response comprising an auxotrophic attenuated strain of
Listeria which expresses an antigen, wherein said auxotrophic
attenuated strain comprises a mutation in both the dal and dat
genes in the genome of said Listeria.
8. The composition of claim 7, wherein said Listeria is L.
monocytogenes.
9. The composition of claim 7, wherein said auxotrophic attenuated
strain further comprises DNA encoding a heterologous antigen.
10. The composition of claim 7, wherein said auxotrophic attenuated
strain further comprises a vector comprising a DNA encoding a
heterologous antigen.
11. The composition of claim 9, wherein said heterologous antigen
is an HIV-1 antigen.
12. The composition of claim 10, wherein said heterologous antigen
is an HIV-1 antigen.
13. An isolated strain of Listeria comprising a mutation in a dal
gene and a mutation in a dat gene which render said strain
auxotrophic for D-alanine.
14. The isolated strain of Listeria of claim 13, further comprising
a heterologous antigen.
Description
FIELD OF THE INVENTION
The invention relates to vaccine vectors comprising bacteria.
BACKGROUND OF THE INVENTION
The use of vaccines is a cost-effective medical tool for the
management of infectious diseases, including infectious diseases
caused by bacteria, viruses, parasites, and fungi. In addition to
effecting protection against infectious diseases, vaccines may now
also be developed which stimulate the host's immune system to
intervene in tumor growth.
Host immune responses include both the humoral immune response
involving antibody production and the cell-mediated immune
response. Protective immunization via vaccine has usually been
designed to induce the formation of humoral antibodies directed
against infectious agents, tumor cells, or the action of toxins.
However, the control of certain diseases characterized by the
presence of tumor cells or by chronic infection of cells with
infectious agents, often requires a cell-mediated immune response
either in place of, or in addition to the generation of antibody.
While the humoral immune response may be induced using live
infectious agents and agents which have been inactivated, a
cellular immune response is most effectively induced through the
use of live agents as vaccines. Such live agents include live
infectious agents which may gain access to the cytoplasm of host
cells where the proteins encoded by these agents are processed into
epitopes which when presented to the cellular immune system, induce
a protective response.
Microorganisms, particularly Salmonella and Shigella which have
been attenuated using a variety of mechanisms, have been examined
for their ability to encode and express heterologous antigens
(Coynault et al., 1996, Mol. Microbiol. 22:149-160; Noriega et al.,
1996, Infect. Immun. 64:3055-3061; Brett et al., 1993, J. Immunol.
150:2869-2884; Fouts et al., 1995, Vaccine 13:1697-1705, Sizemore
et al., 1995, Science 270:299-302). Such bacteria may be useful as
live attenuated bacterial vaccines which serve to induce a cellular
immune response directed against a desired heterologous
antigen.
Listeria monocytogenes (L. monocytogenes) is the prototypic
intracellular bacterial pathogen which elicits a predominantly
cellular immune response when inoculated into an animal (Kaufmann,
1993, Ann. Rev. Immunol. 11:129-163). When used as a vector for the
transmission of genes encoding heterologous antigens derived from
infectious agents or derived from tumor cells, recombinant Listeria
encoding and expressing an appropriate heterologous antigen have
been shown to successfully protect mice against challenge by
lymphocytic choriomeningitis virus (Shen et al., 1995, Proc. Natl.
Acad. Sci. USA 92:3987-3991; Goossens et al., 1995, Int. Immunol.
7:797-802) and influenza virus (Ikonomidis et al., 1997, Vaccine
15:433-440). Further, heterologous antigen expressing recombinant
Listeria have been used to protect mice against lethal tumor cell
challenge (Pan et al., 1995, Nat. Med. 1:471-477; Paterson and
Ikonomidis, 1996, Curr. Opin. Immunol. 8:664-669). In addition, it
is known that a strong cell-mediated immune response directed
against HIV-1 gag protein may be induced in mice infected with a
recombinant L. monocytogenes comprising HIV-1 gag (Frankel et al.,
1995, J. Immunol. 155:4775-4782).
Although the potential broad use of Listeria as a vaccine vector
for the prevention and treatment of infectious disease and cancer
has significant advantages over other vaccines, the issue of safety
during use of Listeria is not trivial. The use of the most common
strain of Listeria, L. monocytogenes, is accompanied by potentially
severe side effects, including the development of listeriosis in
the inoculated animal. This disease, which is normally food-borne,
is characterized by meningitis, septicemia, abortion and often a
high rate of mortality in infected individuals. While natural
infections by L. monocytogenes are fairly rare and may be readily
controlled by a number of antibiotics, the organism may
nevertheless be a serious threat in immunocompromised or pregnant
patients. One large group individuals that might benefit from the
use of L. monocytogenes as a vaccine vector are individuals who are
infected with HIV. However, because these individuals are severely
immunocompromised as a result of their infection, the use of L.
monocytogenes as a vaccine vector is undesirable unless the
bacteria are fully and irreversibly attenuated.
There is a need for the development of a strain of L. monocytogenes
for use as a vaccine in and of itself and for use as a bacterial
vaccine vector which is attenuated to the extent that it is unable
to cause disease in an individual into whom it is inoculated. The
present invention satisfies this need.
SUMMARY OF THE INVENTION
The invention includes a method of eliciting a T cell immune
response to an antigen in a mammal comprising administering to the
mammal an auxotrophic attenuated strain of Listeria which expresses
the antigen, wherein the auxotrophic attenuated strain comprises a
mutation in at least one gene whose protein product is essential
for growth of the Listeria. In a preferred embodiment, the Listeria
is L. monocytogenes. In another preferred embodiment, the
auxotrophic attenuated strain is auxotrophic for the synthesis of
D-alanine. In addition, the mutation comprises a mutation in both
the dal and the dat genes of the Listeria.
In one aspect of the invention, the auxotrophic attenuated strain
further comprises DNA encoding a heterologous antigen, or the the
auxotrophic attenuated strain further comprises a vector comprising
a DNA encoding a heterologous antigen.
The heterologous antigen may be an HIV-1 antigen.
The invention also includes a vaccine comprising an auxotrophic
attenuated strain of Listeria which expresses an antigen, wherein
the auxotrophic attenuated strain comprises a mutation in at least
one gene whose protein product is essential for growth of the
Listeria.
In preferred embodiments, the Listeria is L. monocytogenes. In
other
preferred embodiments, the auxotrophic attenuated strain is
auxotrophic for the synthesis of D-alanine. In yet other preferred
embodiments, the mutation comprises a mutation in both the dal and
the dat genes of the Listeria.
The auxotrophic attenuated strain may further comprise DNA encoding
a heterologous antigen or a vector comprising a DNA encoding a
heterologous antigen.
The heterologous antigen may be an HIV-1 antigen.
Also included in the invention is an isolated nucleic acid sequence
comprising a portion of a Listeria dal gene and an isolated nucleic
acid sequence comprising a portion of a Listeria dat gene.
In addition, the invention includes an isolated strain of Listeria
comprising a mutation in a dal gene and a mutation in a dat gene
which render the strain auxotrophic for D-alanine. In one aspect,
the isolated strain of Listeria further comprises a heterologous
antigen.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1, comprising FIGS. 1A-1C, is the DNA sequence of the L.
monocytogenes alanine racemase gene (dal) of L. monocytogenes (SEQ
ID NO:1) and the amino acid sequence encoded thereby (SEQ ID NO:2).
The lysyl residue involved in the binding of pyridoxal-P is
indicated by an asterisk.
FIG. 2, comprising FIGS. 2A and 2B, depicts the linear alignment of
the deduced amino acid sequences of the alanine racemases of L.
monocytogenes (LMDAL), (SEQ ID NO:2) B. stearothermophilus,
(BSTDAL) (SEQ ID NO:3), and B. subtilis (BSUBDAL) (SEQ ID NO:4).
Identical amino acids are boxed.
FIG. 3, comprising FIGS. 3A-3C, is the DNA sequence of the L.
monocytogenes D-amino acid aminotransferase gene (dat) (SEQ ID
NO:5) and the amino acid sequence encoded thereby (SEQ ID NO:6).
The lysyl residue involved in the binding of pyridoxal-P is
indicated by an asterisk.
FIG. 4, comprising FIGS. 4A and 4B, depicts the linear alignment of
the deduced amino acid sequences of the D-amino acid
aminotransferases of L. monocytogenes (LMDAT), (SEQ ID NO:5) S.
haemolyticus (SHAEDAT), (SEQ ID NO:8) B. sphaericus (BSPHDAT), and
Bacillus species YM-1 (BSPDAT) (SEQ ID NO:9). Identical amino acids
are boxed.
FIG. 5 is a graph depicting the growth requirement for D-alanine of
the dal.sup.- dat.sup.- double mutant strain of L. monocytogenes.
The dal.sup.- dat.sup.- (dal.sup.- dat.sup.-) and wild-type (L.
monocytogenes+) strains of L. monocytogenes were grown in liquid
culture in BHI medium at 37.degree. C. in the presence (+D-ala) or
absence (-D-ala) of exogenous D-alanine (100 .mu.g/ml). In
additional experiments, the mutant cell culture was also provided
D-alanine after 30 minutes and after 60 minutes.
FIG. 6, comprising FIGS. 6A-6C, is a series of images of light
micrographs depicting the growth of wild-type L. monocytogenes
(FIG. 6A) and the dal.sup.- dat.sup.- double mutant strain of L.
monocytogenes (FIG. 6B) in J774 macrophages at 5 hours after
infection with about 5 bacteria per mouse cell. FIG. 6C illustrates
an infection by double mutant bacteria in the continuous presence
of D-alanine (80 .mu.g/ml). Arrowheads point to some mutant
bacteria.
FIG. 7, comprising FIGS. 7A-7C, is a series of graphs depicting
infection of mammalian cells with the dal.sup.- dat.sup.- double
mutant (open circles) and wild-type strains of L. monocytogenes
(closed circles). Mammalian cells which were infected included J774
murine macrophage-like cells (FIG. 7A), primary murine bone marrow
macrophages (FIG. 7B), and human epithelial cells (HeLa) (FIG. 7C).
FIG. 7A also depicts mutant infection in the presence of D-alanine
(100 .mu.g/ml) (closed squares) and in the presence of D-alanine
from 0 to 4 hrs during infection (open squares).
FIG. 8, comprising FIGS. 8A-8E, is a series of images of
photomicrographs depicting the association of actin with
intracytoplasmic wild-type L. monocytogenes (FIG. 8A: 2 hours; FIG.
8B: 5 hours) or with the dal.sup.- dat.sup.- double mutant of L.
monocytogenes (FIG. 8: 2 hours wherein D-alanine was present from 0
to 30 minutes; FIG. 8D: 5 hours, wherein D-alanine was present from
0 to 30 minutes; FIG. 8E: 5 hours, wherein D-alanine was present
continuously), following infection of J744 cells with these
bacteria. The images on the top row illustrate the binding of
FITC-labeled anti-Listerial antibodies to total bacteria, while the
bottom row illustrates the binding of TRITC-labeled phalloidin to
actin. The arrowheads point to some bacteria associated with
actin.
FIG. 9 is a graph depicting the protection of BALB/c mice against
challenge with ten times the LD.sub.50 of wild-type L.
monocytogenes by immunization with the dal.sup.- dat.sup.- double
mutant strain of L. monocytogenes. Groups of 5 mice were immunized
with the following organisms: (1) 4.times.10.sup.2 wild-type L.
monocytogenes, (2) 2.times.10.sup.7 dal.sup.- dat.sup.- (+D-alanine
supplement), (3) 2.times.10.sup.5 dal.sup.- dat.sup.- (+D-alanine
supplement), (4) 2.times.10.sup.4 dal.sup.- dat.sup.- (+D-alanine
supplement), (5) 2.times.10.sup.2 dal.sup.- dat.sup.- mutant
dal.sup.- dat.sup.- (no D-alanine supplement). Mice were challenged
21-28 days after immunization. Log.sub.10 protection was calculated
as described in the Examples.
FIG. 10 is a graph depicting the recovery of bacteria from spleens
of BALB/c mice following sublethal infection with wild type L.
monocytogenes (closed circles), the dal.sup.- dat.sup.- mutant in
the absence of D-alanine (open circles), and the dal.sup.-
dat.sup.- mutant in the presence of 20 mg D-alanine (open squares).
The points at day 0 illustrate the total number of organisms
injected, not the number of bacteria per spleen.
FIG. 11, comprising FIGS. 11A and 11B, is a series of graphs
depicting the cytolytic activity of splenocytes isolated from mice
at 10-14 days after infection with in FIG. 11A, wild type L.
monocytogenes (.circle-solid..smallcircle.), or niave control
(.box-solid..quadrature.). FIG. 11B, dal.sup.- dat.sup.- mutant:
3.times.10.sup.7 bacteria (.DELTA.); 3.times.10.sup.7 bacteria with
boost at 10 days (.DELTA.); 3.times.10.sup.7 bacteria wherein
animals were provided D-alanine subcutaneously
(.circle-solid..smallcircle.); 3.times.10.sup.7 bacteria plus 2
mg/ml D-alanine (.box-solid.) or 0.2 mg/ml D-alanine in drinking
water (.tangle-solidup.).
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to vaccines comprising attenuated
strains of Listeria, wherein the bacteria have been attenuated by
the introduction of auxotrophic mutations in the Listeria genomic
DNA. These strains are herein referred to as attenuated auxotrophic
strains or "AA strains" of Listeria.
It has been discovered in the present invention that the
administration of an AA strain of Listeria to a mammal results in
the development of a host cytotoxic T cell (CTL) response directed
against Listeria following survival of the AA strain in the mammal
for a time sufficient for the development of the response. The AA
strain provides protection against challenge by L. monocytogene and
is therefore suitable for use in a vaccine for protection against
infection by this organism. The AA strain of the invention may thus
be employed as a vaccine for the prevention and/or treatment of
infection by Listeria. In addition, the AA strain of the invention
may have added to it a heterologous gene wherein the gene is
expressed by the AA strain. Such AA strains encoding additional
heterologous genes are useful as bacterial vector vaccines for the
prevention and/or treatment of infection caused by any number of
infectious agents and for the prevention and/or treatment of tumors
in mammals.
AA strains of Listeria that are auxotrophic for D-alanine are
contemplated as part of this invention.
By the term "auxotrophic for D-alanine", as used herein, is meant
that the AA strain of Listeria is unable to synthesize D-alanine in
that it cannot grow in the absence of D-alanine and therefore
requires exogenously added D-alanine for growth.
D-alanine is required for the synthesis of the peptidoglycan
component of the cell wall of virtually all bacteria, and is found
almost exclusively in the microbial world. Wild-type Listeria
species synthesize D-alanine and thus do not require exogenously
added D-alanine for growth. An AA strain of L. monocytogenes has
been discovered in the present invention which is unable to
synthesize D-alanine. This organism may be grown in the laboratory
but is incapable of growth outside the laboratory in unsupplemented
environments, including in the cytoplasm of eukaryotic host cells,
the natural habitat of this organisms during infection. Such
strains of Listeria are useful as vaccines.
By the term "vaccine," as used herein, is meant a population of
bacteria which when inoculated into a mammal has the effect of
stimulating a cellular immune response comprising a T cell
response. The T cell response may be a cytotoxic T cell response
directed against macromolecules produced by the bacteria. However,
the induction of a T cell response comprising other types of T
cells by the vaccine of the invention is also contemplated. For
example, Listeria infection also induces both CD4+ T cells and CD8+
T cells. Induced CD4+ T cells are responsible for the synthesis of
cytokines, such as interferon-.gamma., IL-2 and TNF-.alpha.. CD8+ T
cells may be cytotoxic T cells and also secrete cytokines such as
interferon-.gamma. and TNF-.alpha.. All of these cells and the
molecules synthesized therein play a role in the infection and
subsequent protection of the host against Listeria. Cytokines
produced by these cells activate additional T cells and also
macrophages and recruit polymorphonuclear leukocytes to the site of
infection.
Both prophylactic and therapeutic vaccines are contemplated as
being within the scope of the present invention, that is, vaccines
which are administered either prior to or subsequent to the onset
of disease are included in the invention.
D-alanine auxotrophic mutants useful as vaccine vectors may be
generated in a number of ways. As described in the Examples
presented herein, disruption of one of the alanine racemase gene
(dal) or the D-amino acid aminotransferase gene (dat), each of
which is involved in D-alanine synthesis, did not result in a
bacterial strain which required exogenously added D-alanine for
growth. However, disruption of both the dal gene and the dat gene
generated an AA strain of Listeria which required exogenously added
D-alanine for growth.
The generation of AA strains of Listeria deficient in D-alanine
synthesis may be accomplished in a number of ways that are well
known to those of skill in the art, including deletion mutagenesis,
insertion mutagenesis, and mutagenesis which results in the
generation of frameshift mutations, mutations which effect
premature termination of a protein, or mutation of regulatory
sequences which affect gene expression. Mutagenesis can be
accomplished using recombinant DNA techniques or using traditional
mutagenesis technology using mutagenic chemicals or radiation and
subsequent selection of mutants. Deletion mutants are preferred
because of the accompanying low probability of reversion of the
auxotrophic phenotype. Mutants of D-alanine which are generated
according to the protocols presented herein may be tested for the
ability to grow in the absence of D-alanine in a simple laboratory
culture assay. Those mutants which are unable to grow in the
absence of this compound are selected for further study.
In addition to the aforementioned D-alanine associated genes, other
genes involved in D-alanine synthesis may be used as targets for
mutagenesis of Listeria. Such genes include, but are not limited to
any other known or heretofore unknown D-alanine associated
genes.
Genes which are involved in the synthesis of other metabolic
components in a bacterial cell may also be useful targets for the
generation of attenuated auxotrophic mutants of Listeria, which
mutants may also be capable of serving as bacterial vaccine vectors
for use in the methods of the present invention. The generation and
characterization of such other AA strains of Listeria may be
accomplished in a manner similar to that described herein for the
generation of D-alanine deficient AA strains of Listeria.
Additional potential useful targets for the generation of
additional auxotrophic strains of Listeria include the genes
involved in the synthesis of the cell wall component D-glutamic
acid. To generate D-glutamic acid auxotrophic mutants, it is
necessary to inactivate the dat gene, which is involved in the
conversion of D-glu+pyr to alpha-ketoglutarate+D-ala and the
reverse reaction. It is also necessary to inactivate the glutamate
racemase gene, dga. Other potential useful targets for the
generation of additional auxotrophic strains of Listeria are the
genes involved in the synthesis of diamimopimelic acid. In this
instance, a gene encoding aspartate beta-semialdehyde dehydrogenase
may be inactivated (Sizemore et al., 1995, Science
270:299-302).
By the term "attenuation," as used herein, is meant a diminution in
the ability of the bacterium to cause disease in an animal. In
other words, the pathogenic characteristics of the attenuated
Listeria strain have been lessened compared with wild-type
Listeria, although the attenuated Listeria is capable of growth and
maintenance in culture. Using as an example the intravenous
inoculation of Balb/c mice with an attenuated Listeria, the lethal
dose at which 50% of inoculated animals survive (LD.sub.50) is
preferably increased above the LD.sub.50 of wild-type Listeria by
at least about 10-fold, more preferably by at least about 100-fold,
more preferably at least about 1,000 fold, even more preferably at
least about 10,000 fold, and most preferably at least about
100,000-fold. An attenuated strain of Listeria is thus one which
does not kill an animal to which it is administered, or is one
which kills the animal only when the number of bacteria
administered is vastly greater than the number of wild type
non-attenuated bacteria which would be required to kill the same
animal. An attenuated bacterium should also be construed to mean
one which is incapable of replication in the general environment
because the nutrient required for its growth is not present
therein. Thus, the bacterium is limited to replication in a
controlled environment wherein the required nutrient is provided.
The attenuated strains of the present invention are therefore
environmentally safe in that they are incapable of uncontrolled
replication.
It is believed that any Listeria species capable of infectious
disease may be genetically attenuated according to the methods of
the present invention to yield a useful and safe bacterial vaccine,
provided the attenuated Listeria species exhibits an LD.sub.50 in a
host organism that is significantly greater than that of the
non-attenuated wild type species. Thus, strains of Listeria other
than L. monocytogenes may be used for the generation of attenuated
mutants for use as vaccines. Preferably, the Listeria strain useful
for the generation of attenuated vaccines is L. monocytogenes.
An AA strain of Listeria may be generated which encodes and
expresses a heterologous antigen. The heterologous antigen encoded
by the AA strain of Listeria is one which when expressed by
Listeria is capable of providing protection in an animal against
challenge by the infectious agent from which the heterologous
antigen was derived, or which is capable of affecting tumor growth
and metastasis in a manner which is of benefit to a host organism.
Heterologous antigens which may be introduced into an AA strain of
Listeria by way of DNA encoding the same thus include any antigen
which when expressed by Listeria serves to elicit a cellular immune
response which is of benefit to the host in which the response is
induced. Heterologous antigens therefore include those specified by
infectious agents, wherein an immune response directed against the
antigen serves to prevent or treat disease caused by the agent.
Such heterologous antigens include, but are not limited to, viral,
bacterial, ftmgal or parasite surface proteins and any other
proteins, glycoproteins, lipoprotein, glycolipids, and the like.
Heterologous antigens also include those which provide benefit to a
host organism which is at risk for acquiring or which is diagnosed
as having a tumor. The host organism is preferably a mammal and
most preferably, is a human.
By the term "heterologous antigen," as used herein, is meant a
protein or
peptide, a glycoprotein or glycopeptide, a lipoprotein or
lipopeptide, or any other macromolecule which is not normally
expressed in Listeria, which substantially corresponds to the same
antigen in an infectious agent, a tumor cell or a tumor-related
protein. The heterologous antigen is expressed by an AA strain of
Listeria, and is processed and presented to cytotoxic T-cells upon
infection of mammalian cells by the AA strain. The heterologous
antigen expressed by Listeria species need not precisely match the
corresponding unmodified antigen or protein in the tumor cell or
infectious agent so long as it results in a T-cell response that
recognizes the unmodified antigen or protein which is naturally
expressed in the mammal.
By the term "tumor-related antigen," as used herein, is meant an
antigen which affects tumor growth or metastasis in a host
organism. The tumor-related antigen may be an antigen expressed by
a tumor cell, or it may be an antigen which is expressed by a
non-tumor cell, but which when so expressed, promotes the growth or
metastasis of tumor cells.
The types of tumor antigens and tumor-related antigens which may be
introduced into Listeria by way of incorporating DNA encoding the
same, include any known or heretofore unknown tumor antigen.
The heterologous antigen useful in vaccine development may be
selected using knowledge available to the skilled artisan, and many
antigenic proteins which are expressed by tumor cells or which
affect tumor growth or metastasis or which are expressed by
infectious agents are currently known. For example, viral antigens
which may be considered as useful as heterologous antigens include
but are not limited to the nucleoprotein (NP) of influenza virus
and the gag protein of HIV. Other heterologous antigens include,
but are not limited to, HIV env protein or its component parts
gp120 and gp41, HIV nef protein, and the HIV pol proteins, reverse
transcriptase and protease. In addition, other viral antigens such
as herpesvirus proteins may be useful. The heterologous antigens
need not be limited to being of viral origin. Parasitic antigens,
such as, for example, malerial antigens, are included, as are
funimgal antigens, bacterial antigens and tumor antigens.
As noted herein, a number of proteins expressed by tumor cells are
also known and should be included in the list of heterologous
antigens which may be inserted into the vaccine strain of the
invention. These include, but are not limited to, the bcr/abl
antigen in leukemia, HPVE6 and E7 antigens of the oncogenic virus
associated with cervical cancer, the MAGE1 and MZ2-E antigens in or
associated with melanoma, and the MVC-1 and HER-2 antigens in or
associated with breast cancer.
The introduction of DNA encoding a heterologous antigen into a
strain of Listeria may be accomplished, for example, by the
creation of a recombinant Listeria in which DNA encoding the
heterologous antigen is harbored on a vector, such as a plasmid for
example, which plasmid is maintained and expressed in the Listeria
species. Alternatively, DNA encoding the heterologous antigen may
be stably integrated into the Listeria chromosome by employing, for
example, transposon mutagenesis or by homologous recombination. A
preferred method for producing recombinant Listeria having a gene
encoding a heterologous antigen integrated into the chromosome
thereof, is the induction of homologous recombination between a
temperature sensitive plasmid comprising DNA encoding the antigen
and Listeria chromosomal DNA. Stable transformants of Listeria
which express the desired antigen may be isolated and characterized
as described herein in the experimental examples. This method of
homologous recombination is advantageous in that site directed
insertion of DNA encoding the heterologous antigen is effected,
thereby minimizing the possibility of disruption of other areas of
the Listeria chromosome which may be essential for growth of this
organism.
Several approaches may be employed to express the heterologous
antigen in Listeria species as will be understood by one skilled in
the art once armed with the present disclosure. Genes encoding
heterologous antigens are preferably designed to either facilitate
secretion of the heterologous antigen from the bacterium or to
facilitate expression of the heterologous antigen on the Listeria
cell surface.
While the heterologous antigen preferably comprises only a desired
antigen along with appropriate signal sequences and the like, also
contemplated in the invention is a fusion protein which comprises
the desired heterologous antigen and a secreted or cell surface
protein of Listeria. Listerial proteins which are suitable
components of such fusion proteins include, but are not limited to,
listeriolysin O (LLO) and phosphatidylinositol-specific
phospholipase (PI-PLC). A fusion protein may be generated by
ligating the genes which encode each of the components of the
desired fusion protein, such that both genes are in frame with each
other. Thus, expression of the ligated genes results in a protein
comprising both the heterologous antigen and the listerial protein.
Expression of the ligated genes may be placed under the
transcriptional control of a listerial promoter/regulatory sequence
such that expression of the gene is effected during growth and
replication of the organism. Signal sequences for cell surface
expression and/or secretion of the fused protein may also be added
to genes encoding heterologous antigens in order to effect cell
surface expression and/or secretion of the fused protein.
When the heterologous antigen is used alone (i.e., in the absence
of fused Listeria sequences), it may be advantageous to fuse
thereto signal sequences for cell surface expression and/or
secretion of the heterologous antigen. The procedures for
accomplishing this are well know in the art of bacteriology and
molecular biology.
The DNA encoding the heterologous antigen which is expressed in the
vaccine strain of the invention must be preceeded by a suitable
promoter to facilitate such expression. The appropriate
promoter/regulatory and signal sequences to be used will depend on
the type of listerial protein desired in the fusion protein and
will be readily apparent to those skilled in the art of listeria
molecular biology. For example, preferred L. monocytogenes
promoter/regulatory and/or signal sequences which may be used to
direct expression of a fusion protein include, but are not limited
to, sequences derived from the Listeria hly gene which encodes LLO,
the Listeria p60 gene (Bouwer et al., 1996, Infect. Immun.
64:2515-2522) and possibly the Listeria actA gene which encodes a
surface protein necessary for L. monocytogenes actin assembly.
Other promoter sequences which might be useful in some
circumstances include the plcA gene which encodes PI-PLC, the
listeria mpl gene, which encodes a metalloprotease, the listeria
plcB gene encoding a phospholipase C, and the listeria inlA gene
which encodes internalin, a listeria membrane protein. For a review
of genes involved in L. monocytogenes pathogenesis, see Portnoy et
al. (1992, Infect. and Immun. 60:1263-1267). It is also
contemplated as part of this invention that heterologous regulatory
elements such as promoters derived from phage and promoters or
signal sequences derived from other bacterial species may be
employed for the expression of a heterologous antigen by the
Listeria species.
Examples of the use of recombinant L. monocytogenes strains that
express a heterologous antigen for induction of an immune response
against tumor cell antigens or infectious agent antigens are
described in U.S. patent application Ser. Nos. 08/366,372 and
08/366,477, respectively. The disclosures of these two patent
applications are hereby incorporated herein by reference.
The data presented herein indicate that certain AA strains of
Listeria may undergo osmotic lysis following infection of a host
cell. Thus, if the Listeria which is introduced into the host cell
comprises a vector, the vector is released into the cytoplasm of
the host cell. The vector may comprise DNA encoding a heterologous
antigen. Uptake into the nucleus of the vector DNA enables
transcription of the DNA encoding the heterologous antigen and
subsequent expression of the antigen in and/or secretion of the
same from the infected host cell. Typically, the vector is a
plasmid that is capable of replication in Listeria The vector may
encode a heterologous antigen, wherein expression of the antigen is
under the control of eukaryotic promoter/regulatory sequences.
Typical plasmids having suitable promoters that might be employed
include, but are not limited to, pCMVbeta comprising the immediate
early promoter/enhancer region of human cytomegalovirus, and those
which include the SV40 early promoter region or the mouse mammary
tumor virus LTR promoter region.
Thus, it is also contemplated as part of the present invention that
AA strains of Listeria may be employed as a vaccine for the purpose
of stimulating a CTL immune response against an infectious agent or
a tumor cell, wherein the AA strain comprises a vector encoding a
heterologous antigen that may be expressed using a eukaryotic
expression system. According to the invention, the vector is
propagated in the AA strain of Listeria concomitant with the
propagation of the AA strain itself. The vector may be, for
example, a plasmid that is capable of replication in the AA strain
or the vector may be lysogenic phage. The vector must contain a
prokaryotic origin of replication and must not contain a eukaryotic
origin of replication in order that the vactor is capable of
replication in a prokaryotic cell but, for safety reasons, is
rendered absolutely incapable of replication in eukaryotic
cells.
A cytotoxic T-cell response in a mammal is defined as the
generation of cytotoxic T-cells capable of detectably lysing cells
presenting an antigen against which the T cell response is
directed. Preferably, within the context of the present invention,
the T cell response is directed against a heterologous antigen
expressed in an AA strain of Listeria or which is expressed by a
vector which is delivered to a cell via Listeria infection. Assays
for a cytotoxic T-cell response are well known in the art and
include, for example, a chromium release assay (Frankel et al.,
1995, J. Immunol. 155:4775-4782). In addition to a chromium release
assay, an assay for released lactic acid dehydrogenase may be
performed using a Cytotox 96 kit obtained from Promega Biotech,
WI.
In preferred embodiments and using a chromium release assay, at an
effector cell to target cell ratio of about 50:1, the percentage of
target cell lysis is preferably at least about 10% above the
background level of cell lysis. The background level of cell lysis
is the percent lysis of cells which do not express the target
antigen. More preferably, the percentage of target cell lysis is at
least about 20% above background; more preferably, at least about
40% above background; more preferably, at least about 60% above
background; and most preferably, at least about 70% above
background.
The vaccines of the present invention may be administered to a host
vertebrate animal, preferably a mammal, and more preferably a
human, either alone or in combination with a pharmaceutically
acceptable carrier. The vaccine is administered in an amount
effective to induce an immune response to the Listeria strain
itself or to a heterologous antigen which the Listeria species has
been modified to express. The amount of vaccine to be administered
may be routinely determined by one of skill in the art when in
possession of the present disclosure. A pharmaceutically acceptable
carrier may include, but is not limited to, sterile distilled
water, saline, phosphate buffered solutions or bicarbonate buffered
solutions. The pharmaceutically acceptable carrier selected and the
amount of carrier to be used will depend upon several factors
including the mode of administration, the strain of Listeria and
the age and disease state of the vaccinee. Administration of the
vaccine may be by an oral route, or it may be parenteral,
intranasal, intramuscular, intravascular, intrarectal,
intraperitoneal, or any one of a variety of well-known routes of
administration. The route of administration may be selected in
accordance with the type of infectious agent or tumor to be
treated. The vaccines of the present invention may be administered
in the form of elixirs, capsules or suspensions for oral
administration or in sterile liquids for parenteral or
intravascular administration. The vaccine may also be administered
in conjunction with a suitable adjuvant, which adjuvant will be
readily apparent to the skilled artisan.
The immunogenicity of the AA strain of the invention may be
enhanced in several ways. For example, a booster inoculation
following the initial inoculation may be used to induce an enhanced
CTL response directed against the AA strain.
In another approach, transient suppression of the auxotrophic
phenotype of the AA strain is accomplished by providing the AA
strain with the required nutrient for a period of time shortly
before, after, or concomitant with administration of the Listeria
vaccine to the host. The organism will replicate for the brief
period during which the nutrient is present, after which, upon
exhaustion of the supply of the nutrient, the organism will cease
replication. This brief period of controlled replication will serve
to provide more organisms in the host in a manner similar to that
of natural infection by Listeria, which should stimulate an
enhanced CTL response directed against the organism and antigens
expressed thereby.
In yet another approach, the use of a suicide plasmid may be
employed to conditionally suppress the attenuation of the Listeria
AA strain by temporarily supplying the missing enzyme or enzymes to
the bacterium for synthesis of the essential nutrient. A suitable
suicide plasmid includes pKSV7, the same plasmid which was used to
mediate insertion of genes into the Listeria chromosome as
described herein. This plasmid contains a gram positive (for use in
Listeria), temperature-sensitive replication system such that
growth at 37-40.degree. C. inhibits plasmid replication in
Listeria. This plasmid also contains an E. coli replication system
which is not temperature-sensitive (Smith et al., 1992, Biochimie
74:705-711). The plasmid, or even more temperature-sensitive
derivatives thereof, may be further modified by inserting an
alanine racemase gene into the plasmid, which modified plasmid is
then inserted into an AA strain of Listeria. Listeria cells having
the plasmid inserted therein, are replicated at 30.degree. C. for a
short period of time in order that some molecules of racemase are
accumulated in the cytoplasm. The Listeria cells, so replicated are
then injected into an animal or a human, wherein plasmid
replication then ceases because of the temperature sensitive nature
of the replication system at 37.degree. C. Essentially, the cells
would divide only a few times until the available racemase becomes
diluted out, wherein the cells would cease replication altogether
and become attenuated again. To ensure even tighter temperature
sensitive replication, a temperature sensitive promoter may be used
to regulate expression of the racemase gene and/or temperature
sensitive mutations may be created in the racemase gene itself.
For treatment of cancer, the vaccine of the invention may be used
to protect people at high risk for cancer. In addition, the vaccine
may be used as an immunotherapeutic agent for the treatment of
cancer following debulking of tumor growth by surgery, conventional
chemotherapy, or radiation treatment. Patients receiving such
treatment may be administered a vaccine which expresses a desired
tumor antigen for the purpose of generating a CTL response against
any residual tumor cells in the individual. The vaccine of the
present invention may also be used to inhibit the growth of any
previously established tumors in a human by either eliciting a CTL
response directed against the tumor cells per se, or by eliciting a
CTL response against cells which synthesize tumor promoting
factors, wherein such a CTL response serves to kill those cells
thereby diminishing or ablating the growth of the tumor.
The vaccine of the invention may be maintained in storage until
use. Storage may comprise freezing the vaccine, or maintaining the
vaccine at 4.degree. C., room temperature, or the vaccine may first
be lyophilized and then stored.
The invention particularly contemplates administration of a vaccine
to a human for the purpose of preventing, alleviating, or ablating
HIV infection. The protocol which is described herein for the
administration of a vaccine to a human for the purpose of treating
HIV infection is provided as an example of how to administer an
attenuated auxotrophic Listeria strain as a vaccine to a human.
This protocol should not be construed as being the only protocol
which can be used, but rather, should be construed merely as an
example of the same. Other protocols will become apparent to those
skilled in the art when in possession of the present invention.
Essentially, an auxotrophic strain of L. monocytogenes which
requires D-alanine for growth is constructed as described in the
examples. The mutant is constructed by generating deletion
mutations in both the dal gene and the dat gene, essentially
following the procedures of Camilli et al., (1993, Mol. Microbiol.
8:143-157). The mutant strain is then modified using recombinant
DNA techniques to express an HIV-1 antigen, preferably an antigenic
portion of the gag protein, essentially as described in Frankel et
al. (1995, J. Immunol. 155:4775-4778). A human is then immunized by
injecting a solution containing the auxotrophic L. monocytogenes
strain and a supplement of D-alanine.
One of ordinary skill in the art will know the quantities of cells
and D-alanine which should be administered to the human based upon
a knowledge of the dosages provided herein which are administered
to mice. For example, in BALB/c mice, 10.sup.7 cells and 20 mg of
D-alanine are the preferred dosages. Subsequent injections of the
modified L. monocytogenes cells and D-alanine may also be given to
boost the immune response.
Other HIV-1 antigens or proteins that may be used to generate a
vaccine in accordance with this invention are the HIV env protein
or its component parts, gp120 and gp 41, HIV gag, HIV nef and HIV
pol or its component parts, reverse transcriptase and protease.
Isolated nucleic acid sequences encoding the dal gene and the dat
gene of L. monocytogenes are also contemplated as part of this
invention. In addition to their utility in generating deletion
mutants of L. monocytogenes as disclosed herein, these isolated
nucleic acid sequences encoding the dal gene and the dat gene may
be used as probes and primers in identifying homologous genes in
other Listeria species using PCR and other hybridization technology
available in the art and described, for example, in Sambrook, et
al. (1989, In: Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor Laboratory, New York; Innis et al., ed., 1990, In: PCR
Protocols, Academic Press, Inc., San Diego). Additionally, the
isolated nucleic acid sequences encoding dal or dat may be used to
construct a suicide plasmid that expresses one or both of the
genes. The suicide plasmid(s) may be used to complement the
D-alanine Listeria auxotrophs for a limited time after immunization
as disclosed herein.
An "isolated nucleic acid", as used herein, refers to a nucleic
acid sequence, a DNA or an RNA or fragment thereof which has been
separated from the sequences which flank it in a naturally
occurring state, e.g., a DNA fragment which has been removed from
the sequences which are normally adjacent to the fragment, e.g.,
the sequences adjacent to the fragment in a genome in which it
naturally occurs. The term also applies to nucleic acids which have
been substantially purified from other components which naturally
accompany the nucleic acid, e.g., RNA or DNA or proteins, which
naturally accompany it in the cell. The term therefore includes,
for example, a recombinant DNA which is incorporated into a vector;
into an autonomously replicating plasmid or virus; or into the
genomic DNA of a prokaryote or eukaryote; or which exists as a
separate molecule (e.g, as a cDNA or a genomic or cDNA fragment
produced by PCR amplification, restriction enzyme digestion or
chemical synthesis) independent of other sequences. It also
includes a recombinant DNA which is part of a hybrid gene encoding
additional polypeptide sequence.
Typically probes and primers for use in identifying other dal and
dat genes will comprise a portion of a Listeria dal or dat gene
that is at least about 15 consecutive nucleotides. More typically,
a probe or primer comprises a portion of at least about 20, even
more typically, at least about 30 and even more typically, at least
about 40 consecutive nucleotides of a dal or dat gene of
Listeria.
In other related aspects, the invention includes a vectors which
comprises an isolated nucleic acid encoding dal or dat and which is
preferably capable of directing expression of the protein encoded
by the nucleic acid in a vector-containing cell. The invention
further includes cells comprising a vector encoding dal or dat,
including both prokaryotic and eukaryotic cells.
The isolated nucleic acids of the invention should be construed to
include an RNA or a DNA sequence specifying the dal gene or the dat
gene, and any modified forms thereof, including chemical
modifications of the DNA or RNA which render the nucleotide
sequence more stable when it is cell free or when it is associated
with a cell. Chemical modifications of nucleotides may also be used
to enhance the efficiency with which a nucleotide sequence is taken
up by a cell or the efficiency with which it is expressed in a
cell. Any and all combinations of modifications of the nucleotide
sequences are contemplated in the present invention.
The invention should not be construed as being limited solely to
the DNA and amino acid sequences shown in FIGS. 1 and 3. Once armed
with the present invention, it is readily apparent to one skilled
in the art that any other DNA and encoded amino acid sequence of
the dal and dat genes of other Listeria species may be obtained by
following the procedures described herein. The invention should
therefore be construed to include any and all dal and dat DNA
sequence and corresponding amino acid sequence, having substantial
homology to the dal and dat DNA sequence, and the corresponding
amino acid sequence, shown in FIGS. 1 and 3, respectively (SEQ ID
NOS:1, 2, 5 and 6, respectively). Preferably, DNA which is
substantially homologous is about 50% homologous, more preferably
about 70% homologous, even more preferably about 80% homologous and
most preferably about 90% homologous to the dal or dat DNA sequence
shown in FIGS. 1 and 3, respectively. Preferably, an amino acid
sequence which is substantially homologous is about 50% homologous,
more preferably about 70% homologous, even more preferably about
80% homologous and most preferably about 90% homologous to the
amino acid sequences encoded by the dal and dat genes shown in
FIGS. 1 and 3, respectively (SEQ ID NOS:1, 2, 5 and 6,
respectively).
Homologous as used herein, refers to the subunit sequence
similarity between two polymeric molecules, e.g., between two
nucleic acid molecules, e.g., two DNA molecules or two RNA
molecules, or between two polypeptide molecules. When a subunit
position in both of the two molecules is occupied by the same
monomeric subunit, e.g., if a position in each of two DNA molecules
is occupied by adenine, then they are homologous at that position.
The homology between two sequences is a direct function of the
number of matching or homologous positions, e.g., if half (e.g.,
five positions in a polymer ten subunits in length) of the
positions in two compound sequences are homologous then the two
sequences are 50% homologous, if 90% of the positions, e.g., 9 of
10, are matched or homologous, the two sequences share 90%
homology. By way of example, the DNA sequences 3'ATTGCC5' and
3'TATGCG5' share 50% homology.
The invention is further described in detail by reference to the
following experimental examples. These examples are provided for
purposes of illustration only, and are not intended to be limiting
unless otherwise specified. Thus, the invention should in no way be
construed as being limited to the following examples, but rather,
should be construed to encompass any and all variations which
become evident as a result of the teaching provided herein.
The experimental examples described herein provide procedures and
results which establish that attenuated auxotrophic mutants of L.
monocytogenes are useful as vaccines for eliciting a CTL
response.
Materials and Methods useful in the construction and use of an
attenuated auxotrophic L. monocytogenes strain are now
described.
Bacteria and Plasmids.
The L. monocytogenes strain 10403S (Portnoy et al., 1988, supra)
comprises the wild-type organism used in these studies. This
organism was propagated in brain/heart infusion medium (BHI) (Difco
Labs). L. monocytogenes strain 10403S has an LD.sub.50 of
approximately 3.times.10.sup.4 when injected intravenously or
intraperitoneally into BALB/c mice (Schafer et al., 1992, J.
Immunol. 149:53-59).
E. coli DH5a was used for cloning. This organism was propagated in
Luria broth (Sambrook et al., 1989, supra). The plasmid pKSV7,
which was used for allelic exchange reactions in L. monocytogenes,
is a shuttle vector capable of replication in E. coli, where it is
selected in the presence of 50 .mu.g of ampicillin per ml of media,
and in L. monocytogenes, wherein replication of the plasmid is
temperature sensitive and is selected in the presence of 10 .mu.g
of chloramphenicol per ml of media (Smith et al., 1992, Biochimie
74:705-711). Plasmid DNA obtained from E coli and total DNA
(chromosomal and plasmid) from obtained from Listeria monocytogenes
were isolated using standard methods (Sambrook et al., 1989,
supra).
Identification of D-alanine Synthesis Genes in L. monocytogenes by
Homology with D-alanine Synthesis Genes in Other Gram Positive
Organisms.
Based on sequences of the alanine racemase gene (dal) in
gram-positive organisms (Ferrari et al., 1985, Bio/technology
3:1003-1007; Tanizawa et al., 1988, Biochemistry 27:1311-1316),
primers were designed which corresponded to two 20 base consensus
sequences from highly conserved regions at the 5' and 3' ends of
the dal gene. These primers were modified to reflect the preferred
codon usage in Listeria. These primers were used in a PCR reaction
using chromosomal DNA from either L. monocytogenes or B. subtilis
as templates. A similar sized PCR product (850 nucleotides) was
obtained from both L. monocytogenes and B. subtilis. Analysis of
the 850 nucleotide PCR product from the Listeria template, and the
amino acid sequence encoded thereby, indicated substantial homology
with the alanine racemase genes of the other gram-positive
organisms.
A similar strategy was used to identify and sequence a portion of a
D-amino acid aminotransferase gene (dat) of Listeria, based on
sequences in B. sphaericus, B. species YM-1 (Tanizawa et al., 1989,
supra), and Pucci et al., 1995, J. Bacteriol. 177:336-342). Primers
based on dat sequence in the other gram positive organisms was used
for PCR amplification of L. monocytogenes DNA and a PCR product of
about 400 nucleotides was obtained. Analysis of the DNA sequence of
the 400 nucleotide PCR product, and the amino acid sequence encoded
thereby, indicated substantial homology with the aminotransferase
genes of the other gram positive organisms.
Strategy for Sequence Determination of the Complete Genes.
The sequence of the remaining portions of the L. monocytogenes dal
gene adjoined to the 5' and 3' ends of the central PCR product was
determined using anchored PCR reactions (Rubin et al., 1993, Proc.
Natl. Acad. Sci. USA 90:9280-9284). Briefly, this procedure
utilized a BglII-restriction digest (for the 5' portion of the
gene) or a XbaI digest (for the 3' portion of the gene) of Listeria
chromosomal DNA. The ends of the digested Listeria chromosomal DNA
were then ligated to a small fragment of DNA containing the T7
promoter. A 5'-portion PCR product and a 3'-portion PCR product
were then made and sequenced using primers from within the central
dal gene PCR product and a second primer homologous to the T7
promoter fragment. This procedure permitted determination of the
entire sequence of the dal gene.
The sequence of the remainder of the dat gene was determined by use
of an inverse PCR reaction (Collins et al., 1984, Proc. Natl. Acad.
Sci. USA 81:6812-6816; Triglia et al., 1988, Nucl. Acids Res.
16:8186). Briefly, a HindIII digest of Listeria chromosomal DNA was
permitted to self-ligate under conditions of low DNA concentration
so that mainly single circular molecules would form.
Outward-directing primers with homologies at the two ends of the
original PCR segment of the gene were then used to make a new PCR
product that began at the 5'-end of the original PCR segment,
proceeded to the 5'-end of the gene through the HindIII
self-ligation site and into the 3'-end of the gene. Using this
method, the entire dat gene sequence was obtained.
Production of Mutations in Listeria dal and dat Genes.
The dal gene was inactivated by means of a double allelic exchange
reaction following the protocol of Camilli et al. (Camilli et al.,
1993, Mol. Microbiol 8:143-157). A ts shuttle plasmid pKSV7 (Smith
et al., 1992, supra) construct containing an erythromycin gene
(Shaw and Clewell, 1985, J. Bacteriol. 164:782-796) situated
between a 450-base pair fragment of the 5' end of the 850-base pair
dal gene PCR product and a 450-base pair fragment of the 3' end of
the dal gene PCR product was introduced into Listeria to produce a
double allelic exchange reaction between the chromosomal dal gene
and the plasmid pKSV7 dal construct. A dal deletion mutant covering
about 25% of the gene in the region of its active site was
obtained.
The chromosomal dat gene of L. monocytogenes was also inactivated
using a double allelic exchange reaction. A pKSV7 plasmid construct
containing 450-base pair fragments corresponding to the 5' and 3'
ends of the dat gene PCR product, which had been joined together by
an appropriate PCR reaction, was introduced into Listeria. A double
allelic exchange reaction between the chromosomal dat gene and the
dat plasmid construct resulted in the deletion of 30% of the
central bases of the dat gene.
Infection of Cells in Culture.
To examine the intracellular growth of the attenuated strain of
Listeria in cell culture, monolayers of J774 cells, a murine
macrophage-like cell line, primary murine bone marrow macrophages,
and the human HeLa cell line, were grown on glass coverslips and
infected as described (Portnoy et al., 1988, supra). To enhance the
efficiency of infection of HeLa cells, a naturally non-phagocytic
cell line, the added bacteria were centrifuged onto the HeLa cells
at 543.times.g for 15 minutes. At various times after infection,
samples of the cultures were obtained in order to perform
differential staining for the determination of viable intracellular
bacteria, or for immunohistochemical analysis.
Immunohistochemistry.
Coverslips with attached infected macrophages or HeLa cells were
washed with PBS, and the cells were fixed in 3.2% formalin and
permeabilized using 0.05% Tween 20. Listeria were detected using
rabbit anti-Listeria O antiserum (Difco Laboratories) followed by
LSRSC-labeled donkey anti-rabbit antibodies or coumarin-labeled
goat anti-rabbit antibodies. Actin was detected using FITC- or
TRITC-labeled phalloidin. To distinguish extracellular (or
phagosomal) from intracytoplasmic bacteria, the former were stained
prior to permeabilization of the cells.
Induction of listeriolysin O-specific CTLs.
Female BALB/c mice, 6 to 8 weeks of age (Charles River
Laboratories, Raleigh, NC) were immunized by intraperitoneal
inoculation with either wild-type or dal.sup.- dat.sup.- strains of
L. monocytogenes. After 14 days, some of the mice were boosted with
a second inoculation containing the same number of microorganisms
as were given in the first inoculation. Ten or more days after the
last inoculation, 6.times.10.sup.7 splenocytes obtained from a
given animal were incubated in Iscove's modified DMEM with
3.times.10.sup.7 splenocytes from that same animal that had been
loaded with 10 .mu.M listeriolysin O (LLO) peptide 91-99 during a
60 minute incubation at 37.degree. C. After five days of in vitro
stimulation, the resulting cultures were assayed for the presence
of CTL activity capable of recognizing LLO-peptide-labeled P815
cells following previously published procedures (Wipke et al.,
1993, Eur. J. Immunol. 23:2005-2010; Frankel et al., 1995, supra).
Every determination of lytic activity was corrected for activity in
unlabeled target cells, which exhibited between 1 and 10 percent
lysis.
Animal Protection Studies.
Female BALB/c mice (Bantin-Klingman, Freemont, Calif.) at 8 weeks
of age were immunized with approximately 0.1 LD.sub.50 of viable
wild-type L. monocytogenes or the dal.sup.- dat.sup.- double mutant
strain in 0.2 ml of vehicle, by tail vein injection. Three to four
weeks following immunization, groups of four to five mice each were
challenged with approximately 10 LD.sub.50 of viable wild-type L.
monocytogenes strain 10403 in 0.2 ml of vehicle, by tail vein
injection. Spleens were removed from the mice 48 hours later and
were homogenized individually in 4.5 ml PBS-1% proteose-peptone
using a tissue homogenizer (Tekmar). The homogenates were serially
diluted and plated onto BHI agar. Log.sub.10 protection was
determined by subtracting the mean of the log.sub.10 CFU/spleen
values of the test group from the mean of the log.sub.10 CFU/spleen
values of the normal control group.
Construction of an Auxotrophic Attenuated Strain of L.
monocytogenes Useful as a Vaccine: Construction of an attenuated
strain of L. monocytogenes
Defective in Cell Wall Synthesis
L. monocytogenes was examined to determine whether the bacteria
harbor genes for the synthesis of D-alanine. The alanine racemase
(dal) gene, used by many microorganisms for the synthesis of
D-alanine, has been sequenced in Salmonella (Galakatos et al.,
1986, Biochemistry 25:3255-3260; Wasserman et al., 1984,
Biochemistry 23:5182-5187), B. subtilis (Ferrari et al., 1985,
Bio/technology 3:1003-1007), and B. stearothermophilis (Tanizawa et
al., 1988, Biochemistry 27:1311-1316), but the gene has not been
reported in Listeria. Primers based on the sequences (adjusted for
preferred codon usage in Listeria) of two highly conserved regions
of the dal gene in two different gram-positive organisms were
employed in a PCR reaction performed on L. monocytogenes
chromosomal DNA to search for evidence of the dal gene in Listeria.
A product that exhibited significant homology with the published
dal gene sequences was obtained. The sequence of the remainder of
the L. monocytogenes dal gene was determined as described herein
and is depicted in FIG. 1. The translated protein sequence is
compared with alanine racemases of the other gram-positive
organisms in FIG. 2.
The dal gene was inactivated by an in-frame insertion of a 1.35 kb
fragment of DNA encoding erythromycin resistance at an Spe1 site
near the center of the gene. The resulting dal.sup.- bacteria were
found to grow both in rich bacteriological medium (BHI) as well as
in a synthetic medium in the presence or absence of D-alanine.
Mutation of the dal gene was also achieved by an in-frame deletion
covering 82% of the gene with the same effect.
A second enzyme used by some bacteria for synthesis of D-alanine is
D-amino acid aminotransferase, encoded by the dat gene (Tanizawa et
al., 1989, J. Biol. Chem. 264:2450-2454; Pucci et al., 1995, J.
Bacteriol. 177:336-342). Following the same strategy used to detect
the dal gene in L. monocytogenes, a PCR product that exhibited
significant sequence homology with known dat genes and gene
products was obtained. The sequence obtained from the PCR product
was only the partial gene sequence, and remainder of the dat gene
gene sequence (as depicted in FIG. 3) was determined according to
procedures described herein. The deduced protein sequence of the L.
monocytogenes dat gene is compared with other dat gene products in
FIG. 4.
The L. monocytogenes dat gene was inactivated by in-frame deletion
of 31% of its central region. The growth of the resulting dat.sup.-
bacteria in various bacteriological media was again found to be
independent of the presence of D-alanine.
A double mutant strain of L. monocytogenes, dal.sup.- dat.sup.- -1,
was produced by a double allelic exchange reaction between the
erythromycin-resistant dal.sup.- organism and the shuttle vector
carrying the dat gene deletion. The growth of the double mutant in
bacteriological media was found to be completely dependent on the
presence of D-alanine (FIG. 5). A double mutant containing
deletions in both of the genes, designated dal.sup.- dat.sup.- -12,
had the same phenotype The growth of the double-deletion strain in
the absence of D-alanine could be complemented by a plasmid
carrying the dal gene of B. subtillis. All of the dal.sup.-
dat.sup.- double mutant experiments reported in the following
examples employed the dal.sup.- dat.sup.- -1 double mutant.
Expression of the Defective Phenotype Following Infection of
Eukaryotic Cells
To determine whether a defect in the ability of L. monocytogenes to
synthesize D-alanine would be expressed as an inability to
replicate in the cytoplasm of eukaryotic cells because of the
absence of the required D-alanine in the cytoplasm, several
different cell lines and primary cells in culture were infected
with the wild-type and mutant strains of this organism.
J774 cells are a mouse macrophage-like cell line that readily take
up L. monocytogenes by phagocytosis and permit its cytoplasmic
growth following escape of the bacteria from the phagolysosome
(Tilney et al., 1989, J. Cell Biol. 109:1597-1608). FIG. 6 depicts
typical J774 cells as observed at 5 hours after infection with
about 5 bacteria per cell of either wild-type Listeria (Panel A) or
the double dal.sup.- dat.sup.- mutant Listeria (Panel B). Whereas
large numbers of bacteria were observed to be associated with mouse
cells infected with wild-type Listeria, few were seen following
infection with the double mutant bacteria. Infection by double
mutant bacteria in culture medium containing D-alainine permitted
bacterial growth which was indistinguishable from that seen in
cells infected with wild type Listeria (FIG. 6, Panel C).
Some J774 cells contained small round darkly-staining objects,
often in pairs, that may be spheroblast-like bacteria, although
they were not examined further.
When these cells were infected at higher multiplicities (a
multiplicity of infection of about 1-10), many cells contained
multiple microorganisms, but the double mutant again failed to
multiply. Most double mutant-infected cells possessed pychnotic
nuclei and a pale cytoplasm and presumably were dead; mouse cells
harboring wild-type Listeria did not exhibit this property at any
time after infection.
To quantify some of these observations, the number of intracellular
bacteria (defined by gentamicin resistance) that could form
colonies on medium containing D-alanine was enumerated at several
times after infection (FIG. 7). The data clearly demonstrate that
the double mutant was unable to replicate in J774 cells, and in
fact slowly died during the course of the experiment. The data also
illustrate that the replication-defective phenotype of the double
mutant was supressed by the inclusion of D-alanine (at 100
.mu.g/ml)in the tissue culture medium at the time of infection.
This suppression was reversed within 2 hours after removal of the
D-alanine. The phenotype of the mutant bacteria was also examined
in mouse bone marrow-derived macrophages and in the HeLa cell line
of human epithelaial cells. It was determined that the double
mutant was unable to replicate in either of these cell types as
well (FIG. 7, Panels B and C).
It was again observed that double-mutant-infected macrophages
possessed pychnotic nuclei more frequently than did macrophages
infected with wild-type bacteria. Infection of bone marrow
macrophages was employed to examine the intracytoplasmic status of
the bacteria. Within a few hours after infection of cells by L.
monocytogenes, when the bacteria have escaped from the phagosome,
host actin filaments form a dense cloud around the intracytoplasmic
bacteria, and then rearrange to form a polarized comet tail which
propels the bacteria through the cytoplasm (Tilney et al., 1989,
supra). The actin can readily be visualized using appropriately
labeled anti-Listeria antibodies. At 2 hours post-infection using a
multiplicity of infection of about 5 bacteria per cell, 25% of wild
type bacteria associated with J774 macrophages were surrounded with
a halo of stained actin (FIG. 8, Panel A), and at 5 hours,
virtually 100% of infected cells exhibited actin staining, some
cells having long actin tails (FIG. 8, Panel B). However, the
staining of actin in double-mutant infected macrophages was much
rarer (less than 2%) when compared with wild type infected cells.
Nevertheless, if D-alanine was present during only the 30 minute
period of bacterial adsorption, at 2 hours post-infection '22% of
the mutant cell-associated bacteria were surrounded with actin
(FIG. 8, Panel C); at 5 hours, this number of intracytoplasmic
bacteria had risen to only 27% (FIG. 8, Panel D). If D-alanine was
present during the entire infection period (FIG. 8, Panel E), the
result observed in these cells at 5 hours was indistinguishable
from those observed in wild type infected cells.
Since J774 cells have long been culture adapted and reflect very
few of the normal properties of tissue macrophages, the entry of
mutant bacteria into the cytosol of primary bone marrow macrophages
which had been in culture for only 6 days was examined. Because
these cells demonstrate the high bacterial killing capacity of
normal macrophages, they were infected at a ratio of about 50
bacteria per cell. Under these conditions, at 2 hours after
infection, 6.8% of the double mutant bacteria were found to be
associated with actin in these cells, and this number increased to
the same level as that observed after wild type infection (19%) by
the inclusion of D-alanine for the first 30 minutes of the
infection (1 8.2%) or for the entire period of infection (19.4%).
Therefore, depending on the cell type examined, mutant bacteria in
the absence of D-alanine either exhibited a very low or moderate
efficiency of entering the host cytosol, or exhibited reduced
binding of actin onto their surface. However, the brief presence of
D-alanine during the initial phase of infection allowed a normal
fraction of bacteria to enter the cytosol and bind actin.
Induction of an Immune Response Using the Attenuated Bacteria
Infection of mice with L. monocytogenes produces a long-lived state
of specific immunologic memory that enables the infected host to
resist lethal challenge by the same organism for months following
the primary infection. To determine whether infection of mice with
sub-lethal doses of the double mutant could induce this important
long lasting state of immunity, the following experiments were
performed.
Mice were injected intravenously with 2.times.10.sup.7 (<0.05
LD.sub.50) of the double mutant and were challenged 3 to 4 weeks
later with 10 LD.sub.50 of wild type L. monocytogenes. D-alanine
(20 mg) was provided in the initial inoculum of mutant organisms to
be certain that the organisms were fully viable at the time of
initial infection (this had the effect of reducing the LD.sub.50
about 10 fold). The data presented in FIG. 9 demonstrate that the
level of antilisterial protection was approximately 3 log.sub.10
following a single infection by the mutant bacteria, a similar
level of protection to that generated by immunization with the
wild-type organism. The same dose of mutant bacteria injected
without D-alanine provided little protection.
To determine whether the high degree of protection generated by the
mutant bacteria could be accounted for by their survival and
replication in the infected mice, the spleens of infected animals
were removed and the number of surviving mutant bacteria was
assessed. In FIG. 10 there is shown evidence which indicates that
in the absence of D-alanine, few mutant organisms survived for more
than one day after infection; the presence of D-alanine in the
initial inoculum permitted a few bacteria to survive longer.
Importantly, the almost complete protection obtained using mutant
bacteria occurred in spite of the fact that by 2 days
post-infection more than 100-fold fewer bacteria were detected in
the spleens of mutant infected mice compared with wild type
infected animals.
Listerolysin O peptide 91-99 is the major epitope of the
listerolysin O protein and one of the major epitopes to which mice
respond when mounting a cell mediated immune response against
infection with L. monocytogenes (Bouwer et al., 1996, Infect.
Immun. 64:2515-2522; Harty et al., 1992, J. Exp. Med.
175:1531-1538; Pamer et al., 1991, Nature 353:852-855). To
determine whether the protective immunity generated by infection
with the dal.sup.- dat.sup.- double mutant strain of L.
monocytogenes was associated with the induction of cytolytic T
cells, splenocytes obtained from infected animals were assayed for
their ability to lyse target cells loaded with this peptide. In
FIG. 11 there is shown the fact that animals that were infected
intraperitoneally with 3.times.10.sup.7 and were provided D-alanine
subcutaneously both before and after infection exhibited strong CTL
responses directed against the LLO peptide. Likewise, mice provided
with D-alanine in their drinking water before and after infection
mounted a modest CTL response after single infection with
3.times.10.sup.7 mutant bacteria. In the absence of D-alanine,
animals infected with and boosted one time with 3.times.10.sup.7
bacteria, also exhibited a modest CTL response to LLO peptide
91-99. Single infection with 3.times.10.sup.7 of the double mutant
bacteria in the absence of D-alanine produced no significant
response (FIG. 11).
The disclosures of each and every publication, patent, and patent
application cited herein are hereby incorporated herein by
reference in their entirety.
While this invention has been disclosed with reference to specific
embodiments, it is apparent that other embodiments and variations
of this invention may be devised by others skilled in the art
without departing from the true spirit and scope of the invention.
The appended claims are intended to be construed to include all
such embodiments and equivalent variations.
__________________________________________________________________________
# SEQUENCE LISTING - (1) GENERAL INFORMATION: - (iii) NUMBER OF
SEQUENCES: 9 - (2) INFORMATION FOR SEQ ID NO:1: - (i) SEQUENCE
CHARACTERISTICS: #pairs (A) LENGTH: 1107 base (B) TYPE: nucleic
acid (C) STRANDEDNESS: double (D) TOPOLOGY: linear - (ii) MOLECULE
TYPE: DNA (genomic) - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: -
ATGGTGACAG GCTGGCATCG TCCAACATGG ATTGAAATAG ACCGCGCAGC AA -
#TTCGCGAA 60 - AATATAAAAA ATGAACAAAA TAAACTCCCG GAAAGTGTCG
ACTTATGGGC AG - #TAGTCAAA 120 - GCTAATGCAT ATGGTCACGG AATTATCGAA
GTTGCTAGGA CGGCGAAAGA AG - #CTGGAGCA 180 - AAAGGTTTCT GCGTAGCCAT
TTTAGATGAG GCACTGGCTC TTAGAGAAGC TG - #GATTTCAA 240 - GATGACTTTA
TTCTTGTGCT TGGTGCAACC AGAAAAGAAG ATGCTAATCT GG - #CAGCCAAA 300 -
AACCACATTT CACTTACTGT TTTTAGAGAA GATTGGCTAG AGAATCTAAC GC -
#TAGAAGCA 360 - ACACTTCGAA TTCATTTAAA AGTAGATAGC GGTATGGGGC
GTCTCGGTAT TC - #GTACGACT 420 - GAAGAAGCAC GGCGAATTGA AGCAACCAGT
ACTAATGATC ACCAATTACA AC - #TGGAAGGT 480 - ATTTACACGC ATTTTGCAAC
AGCCGACCAG CTAGAAACTA GTTATTTTGA AC - #AACAATTA 540 - GCTAAGTTCC
AAACGATTTT AACGAGTTTA AAAAAACGAC CAACTTATGT TC - #ATACAGCC 600 -
AATTCAGCTG CTTCATTGTT ACAGCCACAA ATCGGGTTTG ATGCGATTCG CT -
#TTGGTATT 660 - TCGATGTATG GATTAACTCC CTCCACAGAA ATCAAAACTA
GCTTGCCGTT TG - #AGCTTAAA 720 - CCTGCACTTG CACTCTATAC CGAGATGGTT
CATGTGAAAG AACTTGCACC AG - #GCGATAGC 780 - GTTAGCTACG GAGCAACTTA
TACAGCAACA GAGCGAGAAT GGGTTGCGAC AT - #TACCAATT 840 - GGCTATGCGG
ATGGATTGAT TCGTCATTAC AGTGGTTTCC ATGTTTTAGT AG - #ACGGTGAA 900 -
CCAGCTCCAA TCATTGGTCG AGTTTGTATG GATCAAACCA TCATAAAACT AC -
#CACGTGAA 960 - TTTCAAACTG GTTCAAAAGT AACGATAATT GGCAAAGATC
ATGGTAACAC GG - #TAACAGCA 1020 - GATGATGCCG CTCAATATTT AGATACAATT
AATTATGAGG TAACTTGTTT GT - #TAAATGAG 1080 # 1107 ACAT CCATTAG - (2)
INFORMATION FOR SEQ ID NO:2: - (i) SEQUENCE CHARACTERISTICS: #acids
(A) LENGTH: 368 amino (B) TYPE: amino acid (C) STRANDEDNESS: single
(D) TOPOLOGY: linear - (ii) MOLECULE TYPE: protein - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:2: - Met Val Thr Gly Trp His Arg Pro Thr Trp
Il - #e Glu Ile Asp Arg Ala # 15
- Ala Ile Arg Glu Asn Ile Lys Asn Glu Gln As - #n Lys Leu Pro Glu
Ser # 30 - Val Asp Leu Trp Ala Val Val Lys Ala Asn Al - #a Tyr Gly
His Gly Ile # 45 - Ile Glu Val Ala Arg Thr Ala Lys Glu Ala Gl - #y
Ala Lys Gly Phe Cys # 60 - Val Ala Ile Leu Asp Glu Ala Leu Ala Leu
Ar - #g Glu Ala Gly Phe Gln #80 - Asp Asp Phe Ile Leu Val Leu Gly
Ala Thr Ar - #g Lys Glu Asp Ala Asn # 95 - Leu Ala Ala Lys Asn His
Ile Ser Leu Thr Va - #l Phe Arg Glu Asp Trp # 110 - Leu Glu Asn Leu
Thr Leu Glu Ala Thr Leu Ar - #g Ile His Leu Lys Val # 125 - Asp Ser
Gly Met Gly Arg Leu Gly Ile Arg Th - #r Thr Glu Glu Ala Arg # 140 -
Arg Ile Glu Ala Thr Ser Thr Asn Asp His Gl - #n Leu Gln Leu Glu Gly
145 1 - #50 1 - #55 1 - #60 - Ile Tyr Thr His Phe Ala Thr Ala Asp
Gln Le - #u Glu Thr Ser Tyr Phe # 175 - Glu Gln Gln Leu Ala Lys Phe
Gln Thr Ile Le - #u Thr Ser Leu Lys Lys # 190 - Arg Pro Thr Tyr Val
His Thr Ala Asn Ser Al - #a Ala Ser Leu Leu Gln # 205 - Pro Gln Ile
Gly Phe Asp Ala Ile Arg Phe Gl - #y Ile Ser Met Tyr Gly # 220 - Leu
Thr Pro Ser Thr Glu Ile Lys Thr Ser Le - #u Pro Phe Glu Leu Lys 225
2 - #30 2 - #35 2 - #40 - Pro Ala Leu Ala Leu Tyr Thr Glu Met Val
Hi - #s Val Lys Glu Leu Ala # 255 - Pro Gly Asp Ser Val Ser Tyr Gly
Ala Thr Ty - #r Thr Ala Thr Glu Arg # 270 - Glu Trp Val Ala Thr Leu
Pro Ile Gly Tyr Al - #a Asp Gly Leu Ile Arg # 285 - His Tyr Ser Gly
Phe His Val Leu Val Asp Gl - #y Glu Pro Ala Pro Ile # 300 - Ile Gly
Arg Val Cys Met Asp Gln Thr Ile Il - #e Lys Leu Pro Arg Glu 305 3 -
#10 3 - #15 3 - #20 - Phe Gln Thr Gly Ser Lys Val Thr Ile Ile Gl -
#y Lys Asp His Gly Asn # 335 - Thr Val Thr Ala Asp Asp Ala Ala Gln
Tyr Le - #u Asp Thr Ile Asn Tyr # 350 - Glu Val Thr Cys Leu Leu Asn
Glu Arg Ile Pr - #o Arg Lys Tyr Ile His # 365 - (2) INFORMATION FOR
SEQ ID NO:3: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 386
amino (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY:
linear - (ii) MOLECULE TYPE: protein - (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:3: - Met Asn Asp Phe His Arg Asp Thr Trp Ala Gl - #u Val
Asp Leu Asp Ala # 15 - Ile Tyr Asp Asn Val Glu Asn Leu Arg Arg Le -
#u Leu Pro Asp Asp Thr # 30 - His Ile Met Ala Val Val Lys Ala Asn
Ala Ty - #r Gly His Gly Asp Val # 45 - Gln Val Ala Arg Thr Ala Leu
Glu Arg Gly Pr - #o Pro Pro Ala Val Ala # 60 - Phe Leu Asp Glu Ala
Leu Ala Leu Arg Glu Ly - #s Gly Ile Glu Ala Pro #80 - Ile Leu Val
Leu Gly Ala Ser Arg Pro Ala As - #p Ala Ala Leu Ala Ala # 95 - Gln
Gln Arg Ile Ala Leu Thr Val Phe Arg Se - #r Asp Trp Leu Glu Glu #
110 - Ala Ser Ala Leu Tyr Ser Gly Pro Phe Pro Il - #e His Phe His
Leu Lys # 125 - Met Asp Thr Gly Met Gly Arg Leu Gly Val Ly - #s Asp
Glu Glu Glu Thr # 140 - Lys Arg Ile Val Ala Leu Ile Glu Arg His Pr
- #o His Phe Val Leu Glu 145 1 - #50 1 - #55 1 - #60 - Gly Leu Tyr
Thr His Phe Ala Thr Ala Asp Gl - #u Val Asn Thr Asp Tyr # 175 - Phe
Ser Tyr Gln Tyr Thr Arg Phe Leu His Me - #t Leu Glu Trp Leu Pro #
190 - Ser Arg Pro Pro Leu Val His Cys Ala Asn Se - #r Ala Ala Ser
Leu Arg # 205 - Phe Pro Asp Arg Thr Phe Asn Met Val Arg Ph - #e Gly
Ile Ala Met Tyr # 220 - Gly Leu Ala Pro Ser Pro Gly Ile Lys Pro Le
- #u Leu Pro Tyr Pro Leu 225 2 - #30 2 - #35 2 - #40 - Lys Glu Ala
Phe Ser Leu His Ser Arg Leu Va - #l His Val Lys Lys Leu # 255 - Gln
Pro Gly Glu Lys Val Ser Tyr Gly Ala Th - #r Tyr Thr Ala Gln Thr #
270 - Glu Glu Trp Ile Gly Thr Ile Pro Ile Gly Ty - #r Ala Asp Gly
Val Arg # 285 - Arg Leu Gln His Phe His Val Leu Val Asp Gl - #y Gln
Lys Ala Pro Ile # 300 - Val Gly Arg Ile Cys Met Asp Gln Cys Met Il
- #e Arg Leu Pro Gly Pro 305 3 - #10 3 - #15 3 - #20 - Leu Pro Val
Gly Thr Lys Val Thr Leu Ile Gl - #y Arg Gln Gly Asp Glu # 335 - Val
Ile Ser Ile Asp Asp Val Ala Arg His Le - #u Glu Thr Ile Asn Tyr #
350 - Glu Val Pro Cys Thr Ile Ser Tyr Arg Val Pr - #o Arg Ile Phe
Phe Arg # 365 - His Lys Arg Ile Met Glu Val Arg Asn Ala Il - #e Gly
Arg Gly Glu Ser # 380 - Ser Ala 385 - (2) INFORMATION FOR SEQ ID
NO:4: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 389 amino
(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:4: - Met Ser Thr Lys Pro Phe Tyr Arg Asp Thr Tr - #p Ala Glu Ile
Asp Leu # 15 - Ser Ala Ile Lys Glu Asn Val Ser Asn Met Ly - #s Lys
His Ile Gly Glu # 30 - His Val His Leu Met Ala Val Glu Lys Ala As -
#n Ala Tyr Gly His Gly # 45 - Asp Ala Glu Thr Ala Lys Ala Ala Leu
Asp Al - #a Gly Ala Ser Cys Leu # 60 - Ala Met Ala Ile Leu Asp Glu
Ala Ile Ser Le - #u Arg Lys Lys Gly Leu #80 - Lys Ala Pro Ile Leu
Val Leu Gly Ala Val Pr - #o Pro Glu Tyr Val Ala # 95 - Ile Ala Ala
Glu Tyr Asp Val Thr Leu Thr Gl - #y Tyr Ser Val Glu Trp # 110 - Leu
Gln Glu Ala Ala Arg His Thr Lys Lys Gl - #y Ser Leu His Phe His #
125 - Leu Lys Val Asp Thr Gly Met Asn Arg Leu Gl - #y Val Lys Thr
Glu Glu # 140 - Glu Val Gln Asn Val Met Ala Ile Leu Asp Ar - #g Asn
Pro Arg Leu Lys 145 1 - #50 1 - #55 1 - #60 - Cys Lys Gly Val Phe
Thr His Phe Ala Thr Al - #a Asp Glu Lys Glu Arg # 175 - Gly Tyr Phe
Leu Met Gln Phe Glu Arg Phe Ly - #s Glu Leu Ile Ala Pro # 190 - Leu
Pro Leu Lys Asn Leu Met Val His Cys Al - #a Asn Ser Ala Ala Gly #
205 - Leu Arg Leu Lys Lys Gly Phe Phe Asn Ala Va - #l Arg Phe Gly
Ile Gly # 220 - Met Tyr Gly Leu Arg Pro Ser Ala Asp Met Se - #r Asp
Glu Ile Pro Phe 225 2 - #30 2 - #35 2 - #40 - Gln Leu Arg Pro Ala
Phe Thr Leu His Ser Th - #r Leu Ser His Val Lys # 255 - Leu Ile Arg
Lys Gly Glu Ser Val Ser Tyr Gl - #y Ala Glu Tyr Thr Ala # 270 - Glu
Lys Asp Thr Trp Ile Gly Thr Val Pro Va - #l Gly Tyr Ala Asp Gly #
285 - Trp Leu Arg Lys Leu Lys Gly Thr Asp Ile Le - #u Val Lys Gly
Lys Arg # 300 - Leu Lys Ile Ala Gly Arg Ile Cys Met Asp Gl - #n Phe
Met Val Glu Leu 305 3 - #10 3 - #15 3 - #20 - Asp Gln Glu Tyr Pro
Pro Gly Thr Lys Val Th - #r Leu Ile Gly Arg Gln # 335 - Gly Asp Glu
Tyr Ile Ser Met Asp Glu Ile Al - #a Gly Arg Leu Glu Thr # 350 - Ile
Asn Tyr Glu Val Ala Cys Thr Ile Ser Se - #r Arg Val Pro Arg Met #
365 - Phe Leu Glu Asn Gly Ser Ile Met Glu Val Ar - #g Asn Pro Leu
Leu Gln # 380 - Val Asn Ile Ser Asn 385 - (2) INFORMATION FOR SEQ
ID NO:5: - (i) SEQUENCE CHARACTERISTICS: #pairs (A) LENGTH: 870
base (B) TYPE: nucleic acid (C) STRANDEDNESS: double (D) TOPOLOGY:
linear - (ii) MOLECULE TYPE: DNA (genomic) - (xi) SEQUENCE
DESCRIPTION: SEQ ID NO:5: - ATGAAAGTAT TAGTAAATAA CCATTTAGTT
GAAAGAGAAG ATGCCACAGT TG - #ACATTGAA 60 - GACCGCGGAT ATCAGTTTGG
TGATGGTGTA TATGAAGTAG TTCGTCTATA TA - #ATGGAAAA 120 - TTCTTTACTT
ATAATGAACA CATTGATCGC TTATATGCTA GTGCAGCAAA AA - #TTGACTTA 180 -
GTTATTCCTT ATTCCAAAGA AGAGCTACGT GAATTACTTG AAAAATTAGT TG -
#CCGAAAAT 240 - AATATCAATA CAGGGAATGT CTATTTACAA GTGACTCGTG
GTGTTCAAAA CC - #CACGTAAT 300 - CATGTAATCC CTGATGATTT CCCTCTAGAA
GGCGTTTTAA CAGCAGCAGC TC - #GTGAAGTA 360 - CCTAGAAACG AGCGTCAATT
CGTTGAAGGT GGAACGGCGA TTACAGAAGA AG - #ATGTGCGC 420 - TGGTTACGCT
GTGATATTAA GAGCTTAAAC CTTTTAGGAA ATATTCTAGC AA - #AAAATAAA 480 -
GCACATCAAC AAAATGCTTT GGAAGCTATT TTACATCGCG GGGAACAAGT AA -
#CAGAATGT 540 - TCTGCTTCAA ACGTTTCTAT TATTAAAGAT GGTGTATTAT
GGACGCATGC GG - #CAGATAAC 600 - TTAATCTTAA ATGGTATCAC TCGTCAAGTT
ATCATTGATG TTGCGAAAAA GA - #ATGGCATT 660 - CCTGTTAAAG AAGCGGATTT
CACTTTAACA GACCTTCGTG AAGCGGATGA AG - #TGTTCATT 720 - TCAAGTACAA
CTATTGAAAT TACACCTATT ACGCATATTG ACGGAGTTCA AG - #TAGCTGAC 780 -
GGAAAACGTG GACCAATTAC AGCGCAACTT CATCAATATT TTGTAGAAGA AA -
#TCACTCGT 840 # 870 AGTT TGCAAAATAA - (2) INFORMATION FOR SEQ ID
NO:6: - (i) SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 289 amino
(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
- (ii) MOLECULE TYPE: protein - (xi) SEQUENCE DESCRIPTION: SEQ ID
NO:6: - Met Lys Val Leu Val Asn Asn His Leu Val Gl - #u Arg Glu Asp
Ala Thr # 15 - Val Asp Ile Glu Asp Arg Gly Tyr Gln Phe Gl - #y Asp
Gly Val Tyr Glu # 30 - Val Val Arg Leu Tyr Asn Gly Lys Phe Phe Th -
#r Tyr Asn Glu His Ile # 45 - Asp Arg Leu Tyr Ala Ser Ala Ala Lys
Ile As - #p Leu Val Ile Pro Tyr # 60 - Ser Lys Glu Glu Leu Arg Glu
Leu Leu Glu Ly - #s Leu Val Ala Glu Asn #80 - Asn Ile Asn Thr Gly
Asn Val Tyr Leu Gln Va - #l Thr Arg Gly Val Gln # 95 - Asn Pro Arg
Asn His Val Ile Pro Asp Asp Ph - #e Pro Leu Glu Gly Val # 110 - Leu
Thr Ala Ala Ala Arg Glu Val Pro Arg As - #n Glu Arg Gln Phe Val #
125 - Glu Gly Gly Thr Ala Ile Thr Glu Glu Asp Va - #l Arg Trp Leu
Arg Cys # 140 - Asp Ile Lys Ser Leu Asn Leu Leu Gly Asn Il - #e Leu
Ala Lys Asn Lys 145 1 - #50 1 - #55 1 - #60 - Ala His Gln Gln Asn
Ala Leu Glu Ala Ile Le - #u His Arg Gly Glu Gln # 175 - Val Thr Glu
Cys Ser Ala Ser Asn Val Ser Il - #e Ile Lys Asp Gly Val # 190 - Leu
Trp Thr His Ala Ala Asp Asn Leu Ile Le - #u Asn Gly Ile Thr Arg #
205 - Gln Val Ile Ile Asp Val Ala Lys Lys Asn Gl - #y Ile Pro Val
Lys Glu # 220 - Ala Asp Phe Thr Leu Thr Asp Leu Arg Glu Al - #a Asp
Glu Val Phe Ile 225 2 - #30 2 - #35 2 - #40 - Ser Ser Thr Thr Ile
Glu Ile Thr Pro Ile Th - #r His Ile Asp Gly Val # 255 - Gln Val Ala
Asp Gly Lys Arg Gly Pro Ile Th - #r Ala Gln Leu His Gln # 270 - Tyr
Phe Val Glu Glu Ile Thr Arg Ala Cys Gl - #y Glu Leu Glu Phe Ala
# 285 - Lys - (2) INFORMATION FOR SEQ ID NO:7: - (i) SEQUENCE
CHARACTERISTICS: #acids (A) LENGTH: 282 amino (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE:
protein - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: - Met Thr Lys Val
Phe Ile Asn Gly Glu Phe Il - #e Asp Gln Asn Glu Ala # 15 - Lys Val
Ser Tyr Glu Asp Arg Gly Tyr Val Ph - #e Gly Asp Gly Ile Tyr # 30 -
Glu Tyr Ile Arg Ala Tyr Asp Gly Lys Leu Ph - #e Thr Val Thr Glu His
# 45 - Phe Glu Arg Phe Ile Arg Ser Ala Ser Glu Il - #e Gln Leu Asp
Leu Gly # 60 - Tyr Thr Val Glu Glu Leu Ile Asp Val Val Ar - #g Glu
Leu Leu Lys Val #80 - Asn Asn Ile Gln Asn Gly Gly Ile Tyr Ile Gl -
#n Ala Thr Arg Gly Val # 95 - Ala Pro Arg Asn His Ser Phe Pro Thr
Pro Gl - #u Val Lys Pro Val Ile # 110 - Met Ala Phe Ala Lys Ser Tyr
Asp Arg Pro Ty - #r Asp Asp Leu Glu Asn # 125 - Gly Ile Asn Ala Ala
Thr Val Glu Asp Ile Ar - #g Trp Leu Arg Cys Asp # 140 - Ile Lys Ser
Leu Asn Leu Leu Gly Asn Val Le - #u Ala Lys Glu Tyr Ala 145 1 - #50
1 - #55 1 - #60 - Val Lys Tyr Asn Ala Gly Glu Ala Ile Gln Hi - #s
Arg Gly Glu Thr Val # 175 - Thr Glu Gly Ala Ser Ser Asn Val Tyr Ala
Il - #e Lys Asp Gly Ala Ile # 190 - Tyr Thr His Pro Val Asn Asn Tyr
Ile Leu As - #n Gly Ile Thr Arg Lys # 205 - Val Ile Lys Trp Ile Ser
Glu Asp Glu Asp Il - #e Pro Phe Lys Glu Glu # 220 - Thr Phe Thr Val
Glu Phe Leu Lys Asn Ala As - #p Glu Val Ile Val Ser 225 2 - #30 2 -
#35 2 - #40 - Ser Thr Ser Ala Glu Val Thr Pro Val Val Ly - #s Ile
Asp Gly Glu Gln # 255 - Val Gly Asp Gly Lys Val Gly Pro Val Thr Ar
- #g Gln Leu Gln Glu Gly # 270 - Phe Asn Lys Tyr Ile Glu Ser Arg
Ser Ser # 280 - (2) INFORMATION FOR SEQ ID NO:8: - (i) SEQUENCE
CHARACTERISTICS: #acids (A) LENGTH: 283 amino (B) TYPE: amino acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii) MOLECULE TYPE:
protein - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: - Met Ala Tyr Ser
Leu Trp Asn Asp Gln Ile Va - #l Glu Glu Gly Ser Ile # 15 - Thr Ile
Ser Pro Glu Asp Arg Gly Tyr Gln Ph - #e Gly Asp Gly Ile Tyr # 30 -
Glu Val Ile Lys Val Tyr Asn Gly His Met Ph - #e Thr Ala Gln Glu His
# 45 - Ile Asp Arg Phe Tyr Ala Ser Ala Glu Lys Il - #e Arg Leu Val
Ile Pro # 60 - Tyr Thr Lys Asp Val Leu His Lys Leu Leu Hi - #s Asp
Leu Ile Glu Lys #80 - Asn Asn Leu Asn Thr Gly His Val Tyr Phe Gl -
#n Ile Thr Arg Gly Thr # 95 - Thr Ser Arg Asn His Ile Phe Pro Asp
Ala Se - #r Val Pro Ala Val Leu # 110 - Thr Gly Asn Val Lys Thr Gly
Glu Arg Ser Il - #e Glu Asn Phe Glu Lys # 125 - Gly Val Lys Ala Thr
Leu Val Glu Asp Val Ar - #g Trp Leu Arg Cys Asp # 140 - Ile Lys Ser
Leu Asn Leu Leu Gly Ala Val Le - #u Ala Lys Gln Glu Ala 145 1 - #50
1 - #55 1 - #60 - Ser Glu Lys Gly Cys Tyr Glu Ala Ile Leu Hi - #s
Arg Gly Asp Ile Ile # 175 - Thr Glu Cys Ser Ser Ala Asn Val Tyr Gly
Il - #e Lys Asp Gly Lys Leu # 190 - Tyr Thr His Pro Ala Asn Asn Tyr
Ile Leu As - #n Gly Ile Thr Arg Gln # 205 - Val Ile Leu Lys Cys Ala
Ala Glu Ile Asn Le - #u Pro Val Ile Glu Glu # 220 - Pro Met Thr Lys
Gly Asp Leu Leu Thr Met As - #p Glu Ile Ile Val Ser 225 2 - #30 2 -
#35 2 - #40 - Ser Val Ser Ser Glu Val Thr Pro Val Ile As - #p Val
Asp Gly Gln Gln # 255 - Ile Gly Ala Gly Val Pro Gly Glu Trp Thr Ar
- #g Lys Leu Gln Lys Ala # 270 - Phe Glu Ala Lys Leu Pro Ile Ser
Ile Asn Al - #a # 280 - (2) INFORMATION FOR SEQ ID NO:9: - (i)
SEQUENCE CHARACTERISTICS: #acids (A) LENGTH: 283 amino (B) TYPE:
amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear - (ii)
MOLECULE TYPE: protein - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: -
Met Gly Tyr Thr Leu Trp Asn Asp Gln Ile Va - #l Lys Asp Glu Glu Val
# 15 - Lys Ile Asp Lys Glu Asp Arg Gly Tyr Gln Ph - #e Gly Asp Gly
Val Tyr # 30 - Glu Val Val Lys Val Tyr Asn Gly Glu Met Ph - #e Thr
Val Asn Glu His # 45 - Ile Asp Arg Leu Tyr Ala Ser Ala Glu Lys Il -
#e Arg Ile Thr Ile Pro # 60 - Tyr Thr Lys Asp Lys Phe His Gln Leu
Leu Hi - #s Glu Leu Val Glu Lys #80 - Asn Glu Leu Asn Thr Gly His
Ile Tyr Phe Gl - #n Val Thr Arg Gly Thr # 95 - Ser Pro Arg Ala His
Gln Phe Pro Glu Asn Th - #r Val Lys Pro Val Ile # 110 - Ile Gly Tyr
Thr Lys Glu Asn Pro Arg Pro Le - #u Glu Asn Leu Glu Lys # 125 - Gly
Val Lys Ala Thr Phe Val Glu Asp Ile Ar - #g Trp Leu Arg Cys Asp #
140 - Ile Lys Ser Leu Asn Leu Leu Gly Ala Val Le - #u Ala Lys Gln
Glu Ala 145 1 - #50 1 - #55 1 - #60 - His Glu Lys Gly Cys Tyr Glu
Ala Ile Leu Hi - #s Arg Asn Asn Thr Val # 175 - Thr Glu Gly Ser Ser
Ser Asn Val Phe Gly Il - #e Lys Asp Gly Ile Leu # 190 - Tyr Thr His
Pro Ala Asn Asn Met Ile Leu Ly - #s Gly Ile Thr Arg Asp # 205 - Val
Val Ile Ala Cys Ala Asn Glu Ile Asn Me - #t Pro Val Lys Glu Ile #
220 - Pro Phe Thr Thr His Glu Ala Leu Lys Met As - #p Glu Leu Phe
Val Thr 225 2 - #30 2 - #35 2 - #40 - Ser Thr Thr Ser Glu Ile Thr
Pro Val Ile Gl - #u Ile Asp Gly Lys Leu # 255 - Ile Arg Asp Gly Lys
Val Gly Glu Trp Thr Ar - #g Lys Leu Gln Lys Gln # 270 - Phe Glu Thr
Lys Ile Pro Lys Pro Leu His Il - #e # 280
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